1. Field of the Invention
The invention relates to making targeted nanoparticle delivery systems for drugs, magnets, and sensors. The invention relates to the preparation of microemulsion precursors whereby the dispersed droplets are templates for the curing of solid nanoparticles. The invention also relates to making solid nanoparticles from microemulsion precursors without the use of light, electricity, free-radicals, or γ-rays to make nanoparticles. The invention also relates to a nanoparticulate delivery system for delivering a molecule of interest to the body.
2. Brief Description of the Related Art
Nanotechnology is becoming increasingly more important in the pharmaceutical, chemical and engineering fields. This is primarily due to the fact that particles made at the nanoscale have much different physical, chemical, and biological properties than larger particles. For example, in the pharmaceutical field, nanoparticles have been used to more efficiently deliver drugs, genes, diagnostics, and vaccines (Douglas et al., 1987; MacLaughlin, et al., 1998; Kreuter 1995). Due to their small size, nanoparticles can aid in the direct entry of entrapped molecules into cells (either non-specifically or specifically via cell targeting ligands). Cellular uptake of drug molecules is often desirable and even necessary if the mechanism of action of the drug requires it to be in the cell as in the case of larger biologically-based molecules such as antisense oligonucleotides, ribozymes, and plasmid DNA. Further, the benefit of being able to deliver a vaccine antigen intracellularly to achieve a cellular-based immune response has been established (for a review see Mumper et al., 1999). However, getting these larger molecules efficiently into cells is difficult. Unlike small drugs, which may efficiently enter cells by diffusion and/or transport mechanisms, large molecules often require a carrier system to achieve sufficiently high intracellular concentrations. Nanoparticles may provide a way of increasing the cellular uptake of larger molecules if these molecules can be efficiently packaged into pharmaceutically acceptable carriers using a cost-effective method.
Gene therapy has emerged as a promising approach for the treatment of a number of genetic and acquired diseases. Non-viral gene therapy involves the delivery of genetic material (plasmid DNA) into cells of the body to produce therapeutic proteins endogenously by exploiting the cell's transcriptional and translational machinery. Most non-viral gene delivery strategies employ polyelectrolyte complexation using cationic lipids, peptides, or polymers to complex and condense negatively charged plasmid DNA into particles having diameters in the 100–1000 nm range. The complexation strategy is fraught with problems since: i) the cationic molecules are relatively toxic materials and are not approved by the FDA in any marketed medical product, ii) the complexes are prone to aggregation at or near charge neutrality, iii) stable particles having diameters below 100 nm are very difficult to engineer, iv) scale-up of these complexes is complicated and expensive since very controlled mixing systems are needed to introduce the ions in solution, and v) the complexes tend to aggregate or dissociate when injected in the body.
Although a few reports in the literature have demonstrated proof-of-concept in animals, attempts to specifically target polyelectrolyte complexes to cells in the body using cell-specific ligands have been largely unsuccessful. Also, no such technology has advanced to clinical testing in humans. Contributing factors to this lack of success may be that these ligands (i.e., monoclonal antibodies, carbohydrates, etc.) are attached to biologically unstable particles and/or that the these particles cannot be made small enough to be efficiently taken up by cells by receptor-mediated endocytosis.
As an alternative to polyelectrolyte complexation, researchers have also attempted to encapsulate plasmid DNA into conventional solid nanoparticles based on biodegradable polymers such as polylactic acid-co-glycolic acid (Ando et al., 1999; Wang et al., 1999), gelatin (Truong-Le, et al., 1998), and other polymers (Mumper and Klakamp, 1999). However, these techniques and systems have disadvantages such as: a) the relatively high cost of these carrier materials, b) the unknown safety of some of these materials, c) the use of rigorous processes typically used to make the nanoparticles (i.e., interfacial polymerization and/or high-torque mechanical mixing that may be damaging to biologically-based drugs and expensive to scale-up and manufacture), d) the inability to produce nanoparticles below 50 nm, and e) the low encapsulation efficiency of plasmid DNA.
Yet another alternative to polyelectrolyte complexation is to incorporate plasmid DNA into microemulsions. A microemulsion is a stable biphasic mixture of two immiscible liquids stabilized by a surfactant and usually a co-surfactant. Microemulsions are thermodynamically stable, isotropically clear, form without excessive mixing, and have dispersed droplets in the range of 5 nm to 100 nm diameter. Microemulsions have been proposed as drug delivery systems to enhance the absorption of drugs across biological membranes (Bhargava et al. 1987; Ho et al. 1996; Constantinides, 1995). Although microemulsions have advantages as delivery systems, they do have important limitations. For example, the dispersed droplets are a liquid and are not stable in biological fluids. Thus, microemulsions are not effective in delivering drugs intracellularly or targeting drugs to different cells in the body.
A significant advancement in the field of non-viral gene delivery would be made if one could avoid the problems associated with polyelectrolyte complexation and instead combine the unique advantages of solid nanoparticles and microemulsions into one pharmaceutically engineered gene delivery system.
Finally, there have been a handful of reports pertaining to the use of microemulsions to make nanoparticles (Li et al., 1999; Cavalli et al., 1999; Bocca et al., 1998; Tojo et al., 1998; Munshi et al., 1997; Ruys et al., 1999). These reports have primarily dealt with the preparation of water-in-oil (hydrocarbon) microemulsions (Lade et al., 2000; Song et al., 2000; Porta et al., 1999) whereby nanoparticles are formed in the water phase by the use of photochemistry (Agostiano et al., 2000), γ-rays (Xiangling et al., 1999), or electrochemistry (Tang et al., 2000) to induce crosslinking, polymerization (Fang et al., 2000; Capek, 1999; Meier, 1999) and/or complexation of the appropriate agents in the water phase. The great majority of these reports do not use pharmaceutically acceptable materials or methods of preparation that would be suitable for scale-up and preparation of nanoparticles containing drugs, magnets, or sensors that are intended for use in humans.
U.S. Pat. No. 4,826,689 to Violanto, discloses methods of making uniformly sized particles of less than 10 microns from water-insoluble drugs by precipitation. Although Violanto discloses a method of making drug particles by precipitation, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-Water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Pat. No. 4,997,599 to Steiner, discloses the preparation of cellulose acetate microspheres having a size of less than 1 micron to a maximum of 1000 microns by spraying a polymer solution through a nozzle. Although Steiner discloses the use of a film-forming cellulose polymer, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Pat. No. 5,049,322 to Devissaguet discloses a process of preparing a colloidal system containing nanocapsules of less than about 500 nanometers. The patent reports that the colloidal system of nanocapsules forms practically instantaneously with gentle agitation. The wall of the nanoparticles is reported to be preferably formed of a film forming polymer, e.g., cellulose, and the core may be a biologically active substance. Although the patent describes nano-sized products, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Pat. No. 5,500,224 to Vranckx describes pharmaceutical compositions containing nanocapsules. The nanocapsules are prepared by adding an aqueous solution containing an active ingredient to an oil to form a water-in-oil emulsion and removing the nanocapsules having a size of less than 500 nanometers. Although the patent describes nano-sized products, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Pat. No. 5,733,526 to Trevino discloses hydrocarbon oil/fluorochemical preparations which may be used for the administration of bioactive agents. It is reported that the hydrocarbon oil, e.g., paraffin or vegetable oil, is preferably dispersed in a continuous fluorochemical phase. In an embodiment, the patent discloses a hydrocarbon oil-fluorochemical disperse phase in a continuous polar liquid. Trevino does not appear to disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions wherein the dispersed alcohol or liquid hydrocarbon phases contain a film-forming substance dissolved or dispersed therein. Further, the patent does not disclose the use of such microemulsions to prepare solid nanoparticles containing drug.
U.S. Pat. No. 5,250,236 by Gasco describes the use of solid lipid microspheres that are formed by diluting one volume of the mixture of molten lipid substance, water, surfactant and possibly a co-surfactant to 100 volumes of cold water. Gasco teaches the preparation of microspheres smaller than one micrometer and in particular between 50–800 nanometers, and preferably between 100 and 400 nanometers. Gasco also teaches the preparation of microspheres wherein said solid lipid microspheres may contain a pharmacologically active substance, such as a drug. Gasco does not teach the use of nanoparticles made from oil-in-water microemulsion precursors wherein said nanoparticles containing drugs are formed directly by cooling the oil-in-water microemulsion with no dilution of the most useful system.
Conventional microemulsions are water-in-oil type, and use various methods of curing the nanoparticles (i.e., crosslinking, polymerization, radiation, and so on). There is a need in the art to provide a non water-in-oil type microemulsions using curing methods specific to those non water-in-oil microemulsions, such as by cooling and evaporation or by the addition of a solvent, to prepare solid nanoparticles containing drug or other molecules of interest. An additional advantage of this invention over prior art is that the described nanoparticle systems can be engineered rapidly, reproducibly, and cost-effectively from the microemulsion precursors in a one-step process and contained in one manufacturing vessel, vial, or container.